This site needs JavaScript to work properly. Please enable it to take advantage of the complete set of features!
Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log in
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jan 3;12(1):e0166903.
doi: 10.1371/journal.pone.0166903. eCollection 2017.

Recombinase Polymerase Amplification Assay-A Simple, Fast and Cost-Effective Alternative to Real Time PCR for Specific Detection of Feline Herpesvirus-1

Affiliations

Recombinase Polymerase Amplification Assay-A Simple, Fast and Cost-Effective Alternative to Real Time PCR for Specific Detection of Feline Herpesvirus-1

Jianchang Wang et al. PLoS One. .

Abstract

Feline herpesvirus 1 (FHV-1), an enveloped dsDNA virus, is one of the major pathogens of feline upper respiratory tract disease (URTD) and ocular disease. Currently, polymerase chain reaction (PCR) remains the gold standard diagnostic tool for FHV-1 infection but is relatively expensive, requires well-equipped laboratories and is not suitable for field tests. Recombinase polymerase amplification (RPA), an isothermal gene amplification technology, has been explored for the molecular diagnosis of infectious diseases. In this study, an exo-RPA assay for FHV-1 detection was developed and validated. Primers targeting specifically the thymidine kinase (TK) gene of FHV-1 were designed. The RPA reaction was performed successfully at 39°C and the results were obtained within 20 min. Using different copy numbers of recombinant plasmid DNA that contains the TK gene as template, we showed the detection limit of exo-RPA was 102 copies DNA/reaction, the same as that of real time PCR. The exo-RPA assay did not cross-detect feline panleukopenia virus, feline calicivirus, bovine herpesvirus-1, pseudorabies virus or chlamydia psittaci, a panel of pathogens important in feline URTD or other viruses in Alphaherpesvirinae, demonstrating high specificity. The assay was validated by testing 120 nasal and ocular conjunctival swabs of cats, and the results were compared with those obtained with real-time PCR. Both assays provided the same testing results in the clinical samples. Compared with real time PCR, the exo-RPA assay uses less-complex equipment that is portable and the reaction is completed much faster. Additionally, commercial RPA reagents in vacuum-sealed pouches can tolerate temperatures up to room temperature for days without loss of activity, suitable for shipment and storage for field tests. Taken together, the exo-RPA assay is a simple, fast and cost-effective alternative to real time PCR, suitable for use in less advanced laboratories and for field detection of FHV-1 infection.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Specificity analysis of the exo-RPA assay.
As shown in this figure, only FHV-1 DNA was positively amplified (curve 1). In contrast, feline calicivirus, chlamydia psittaci and other viruses commonly seen in cats, i.e., FPV, BHV-1, PRV, were all tested negative (curves 2–6, respectively). Curve 7 used nuclease-free water as a negative control. Shown in this figure is one representative plot out of 5 independent reactions.
Fig 2
Fig 2. Sensitivity analysis of the exo-RPA assay.
Different copy numbers of plasmid pMD19-T-TK DNA (106 to 100 copies) were amplified by either RPA reactions or real time PCR. As shown in this figure, the detection limit was 102 copies of DNA/reaction for both the exo-RPA assay (panel A) and real time PCR (panel B). The copy numbers used as template for curve 1–7 were 106, 105, 104, 103, 102, 101 and 100, respectively. Shown in this figure is one representative plot out of 5 independent reactions for RPA and real time PCR, respectively.

References

    1. Gaskell R, Dawson S, Radford A, Thiry E. Feline herpesvirus. Vet Res. 2007; 38: 337–354. 10.1051/vetres:2006063 - DOI - PubMed
    1. Townsend WM, Jacobi S, Tai SH, Kiupel M, Wise AG, Maes RK. Ocular and neural distribution of feline herpesvirus-1 during active and latent experimental infection in cats. BMC Vet Res. 2013; 9:185 10.1186/1746-6148年9月18日5 - DOI - PMC - PubMed
    1. Maggs DJ, Lappin MR, Reif JS, Collins JK, Carman J, Dawson DA, Bruns C. Evaluation of serologic and viral detection methods for diagnosing feline herpesvirus-1 infection in cats with acute respiratory tract or chronic ocular disease. J Am Vet Med Assoc. 1999; 214:502–507. - PubMed
    1. Zicola A, Saegerman C, Quatpers D, Viandier J, Thiry E. Feline herpesvirus 1 and feline calicivirus infections in a heterogeneous cat population of a rescue shelter. J Feline Med Surg. 2009; 11: 1023–1027. 10.1016/j.jfms.2009年05月02日3 - DOI - PMC - PubMed
    1. Sandmeyer LS, Waldner CL, Bauer BS, Wen X, Bienzle D. Comparison of polymerase chain reaction tests for diagnosis of feline herpesvirus, Chlamydophila felis, and Mycoplasma spp. infection in cats with ocular disease in Canada. Can Vet J. 2010; 51: 629–633. - PMC - PubMed

Publication types

MeSH terms

Full text links
Public Library of Science full text link Public Library of Science Free PMC article
Cite

AltStyle によって変換されたページ (->オリジナル) /